Project Summary/Abstract Our goal is to investigate the functions and mechanisms of N6-methyladenosine (m6A) modification of HIV-1 RNA during viral infection. The internal m6A modification of cellular RNAs is a novel mechanism of post-transcriptional control of gene expression, which is coordinately regulated by three groups of host proteins, including methyltransferases (writers), demethylases (erasers), and m6A-selective- binding proteins (readers). Binding of m6A-modified cellular RNA by the readers significantly affects various aspects of RNA functions during translation. Three recent publications, including one from our group, highlighted the critical role of m6A modification of HIV-1 RNA in regulating viral replication. However, these studies reported some different results and suggested distinct mechanisms. To clarify the discrepancy, it is essential to define the mechanisms by which m6A modification of HIV-1 RNA regulates viral replication in primary CD4+ T-cells. We identified multiple regions of m6A modification in HIV-1 RNA bound by the readers in HIV-1- infected primary CD4+ T-cells. We found that the expression levels of the readers significantly modulated HIV-1 infection in target cells. Knockdown of the writers or erasers in virus-producing cells significantly affected HIV-1 Gag protein synthesis, suggesting the importance of m6A modification of HIV-1 RNA in regulating viral gene expression. Interestingly, we observed that HIV-1 infection upregulated the expression of endogenous readers in primary CD4+ T-cells. Our central hypothesize is that reversible m6A modification of HIV-1 RNA regulates viral replication in CD4+ T- cells by affecting the structure, stability, splicing, and/or trafficking of HIV-1 RNA. We will test this hypothesis in three specific aims using interdisciplinary approaches. Aim 1. To identify the m6A residues in HIV-1 RNA and to investigate the effects of m6A modification on HIV-1 RNA structure and interactions with the reader proteins; Aim 2. To investigate the effects of m6A modification on the stability, splicing and trafficking of HIV-1 RNA in infected primary CD4+ T-cells; and Aim 3. To investigate the effects of HIV-1 infection and proinflammatory cytokines on the expression and localization of the readers, writers, and erasers and to define the underlying mechanisms. Overall impact: Accomplishing our proposed studies will define the mechanisms by which HIV-1 RNA m6A modification regulates viral infection in CD4+ T-cells. Investigations of the HIV-1 RNA m6A modification and interactions with host proteins represent a new area of HIV-1 RNA biology, which can facilitate therapeutic development against HIV-1 infection.